The present invention relates generally to the purification of proteins produced by recombinant DNA technologies. More particularly, it concerns the clarification of protein suspensions containing soluble and insoluble components via flocculation with anionic polymers.
The use of flocculating agents has been described in several industrial settings, including the biotechnology industry. For example, U.S. Pat. No. 5,047,511 describes the use of cationic flocculating agents in the recovery of recombinant somatotropin protein from a protein solution containing soluble high molecular weight contaminating proteins. This involved the selective precipitation of contaminating high molecular weight proteins by adding a cationic flocculant containing quaternary ammonium groups and then recovering the somatotropin from the solution.
Somatotropins, also known as growth hormones, are polypeptides produced and secreted by cells of the pituitary gland. These proteins are known to be effective in promoting pre-adult skeletal growth and meat production of beef cattle and swine, and can be produced reliably and inexpensively in large quantities by recombinant DNA technology. In addition, they are known to affect a variety of metabolic processes including the stimulation of lactation, improvements of the efficiency of converting feed to meat or milk, lipid-mobilizing effects, and others.
Recombinant DNA technology provides a means for the large scale production of heterologous proteins of interest in bacterial host cells. In the case of somatotropin, a growth hormone, the protein is sequestered in inclusion bodies within the cytoplasm of the host cells. The inclusion bodies can be recovered from the host cell culture by disrupting the cell so as to release the inclusion bodies, and thereafter collecting the inclusion bodies as a solid pellet by differential centrifugation. The inclusion bodies are solubilized in an aqueous solution of a suitable chaotropic agent such as urea or guanidine hydrochloride at an alkaline pH and subsequently naturized by contact with a mild oxidizing agent to form intramolecular disulfide bonds and to refold the protein into its biologically active, native conformation. Methods for the solubilization and naturation of somatotropin protein produced by E. coli bacteria are described in U.S. Pat. No. 4,511,502 and U.S. Pat. No. 4,652,630, each of which is incorporated herein by reference.
The somatotropin refold solution obtained from the naturation step (as described for example in U.S. Pat. Nos. 4,511,502 and 4,652,630) comprises an aqueous solution of somatotropin monomers, dimers and higher oligomers, along with residue and other debris from the host cells. Of these, the somatotropin monomer is the desired biologically active agent. U.S. Pat. No. 5,182,369, the disclosure of which is incorporated herein by reference, describes the selective precipitation of somatotropin dimer and higher oligomers together with residual host cell proteins and other contaminating substances from a pH-adjusted somatotropin refold solution, leaving the desired somatotropin monomer as the primary soluble constituent of the suspension.
Once the somatotropin oligomers and other contaminants have been selectively precipitated using this approach, it is necessary to remove the precipitated proteins and other insoluble contaminants from the suspension in order to obtain somatotropin monomers of the desired purity. Such liquid/solid separations as those required for this purification step are employed in most industrial biotechnological processes and are frequently accomplished via centrifugation and/or filtration procedures.
Flocculating agents can be employed to improve liquid/solid separations by aggregating the solids that are present in a protein suspension, thereby increasing the particle size of the solids (for review, see Halverson and Panzer, 1980). An increase in particle size is particularly beneficial in centrifugation and sedimentation applications where the particle sedimentation velocity is proportional to the square of the particle radius. The increased sedimentation velocity that results from larger particle sizes can improve productivity in any type of liquid/solid separation where particle sedimentation velocity is a factor.